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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In an OECD 421 study (Robinson, 2018), groups of 10 male and female Han Wistar rats were exposed nose-only by inhalation, 6 hours per day, 7 days per week, for 2 weeks pre-pairing up to Day 19 post-coitum (pc) (a minimum of 4 weeks for the males) and from Day 5 of lactation until Day 12 to a respirable aerosol of Graphistrength C100 at actual concentrations of 0, 0.285, 1.41 and 5.60 mg/m3 (MMAD 2.4-3.0 µm, GSD 2.38). The animals were checked twice daily for mortality, morbidity and clinical signs during the treatment period. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 3, 7, 10, 14 and 20 post-coitum (p.c.)and 1-3, 4-6 and 7-12 of lactation. Estrous cyclicity was checked for 15 days before pairing. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1, 4, 7 and 13 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 4 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p. Litter standardization was performed on Day 4 p.p.in order to reduce the litter size-induced variability in pup growth and development. Thyroid hormone (TSH, T3 and T4) plasma levels were determined on the day of sacrifice in parental males and in Day 13 p.p. pups. Final body weights and selected organs weights (lungs, ovaries and male reproductive organs) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on larynx, lungs, tracheobronchial lymph nodes in all groups and on nasal passages, ovaries (with oviducts), epididymides and/or testes, pituitary in the control and high-dose groups, and on all macroscopic lesions. A macroscopic post-mortem examination was performed on pups on Day 14 p.p., including those found dead before study termination.

Test item (black) staining on the head was recorded as a sign associated with dosing for all test groups, the incidence of this observation was related to exposure concentration. Wet fur seen on occasion is considered to be associated with the method of restraint. Test item (black) staining was also noted during the detailed weekly physical examinations on the head and forelimbs. No body weight gain was observed for females exposed to 5.60 mg/m3 compared with control over the first 2 weeks of exposures. During the gestation phase body weight gains for treated females were similar to control, during the lactation phase lower body weight gains were observed for all treated female groups, as low as 0.66X control; however this finding lacked exposure relationship and is thus considered to be unrelated to the test item. There were no effects on body weight gain for males. Food consumption was slightly low for females exposed to 1.41 or 5.60 mg/m3 over the first 2 weeks of exposures compared with control (approx. 0.9X control). Food consumption during gestation was similar to control for all treated female groups but was slightly lower than control for females exposed to 1.41 or 5.60 mg/m3 during lactation (0.9X control). There were no effects on food consumption for males. There were no test item related effects on mating performance, estrous cycles, pre-coital Interval, fertility and gestation length, and estrous stage at termination. All thyroid hormone sampling were completed, T4 results from Day 13 pups and adult males are pending. There were no effects on the number of implantations, litter size, sex ratio, survival indices or ano-genital distances. Group mean body weight change for male offspring from the 5.60 mg/m3 group was slightly higher than the control group (1.1X) but in the absence of similar effects in females and the small magnitude of the change, was considered unrelated to the test item. Nipple counts on Day 13 were zero for all male offspring. Group mean lung/bronchi absolute weights (unadjusted and adjusted) were higher than control for both sexes exposed to 1.41 or 5.60 mg/m3 , up to 1.3X for adjusted weights, for males and females (exposure related). Abnormal color (grey) was seen in the lungs of all animals exposed to 5.60 mg/m3 and in the majority of animals exposed to 1.41 mg/m3, in both sexes. Abnormal color (grey) was seen in the tracheobronchial lymph nodes of all females exposed to 5.60 mg/m3 and the majority of females exposed to 1.41 mg/m3. Dark lymph nodes were noted in some males exposed to 1.41 or 5.60 mg/m3. Enlargement was seen in the majority of males exposed to 5.60 mg/m3 and several males exposed to 1.41 mg/m3. Changes related to inhalation administration of the test item were seen in the larynx of females only and lungs of both sexes. In the larynx, minimal squamous metaplasia occurred in treated females, with an exposure- related increase in incidence. This finding was characterised by a focal loss of cilia and focal flattening of the surface epithelial cells, primarily at the basis of the epiglottis. This change is indicative of low grade irritation of the epithelium of the ventral larynx and is considered non-adverse. The rat larynx is highly sensitive to irritant materials due to species-specific anatomical, airflow and histological features, and as such squamous metaplasia in this region is generally considered irrelevant to man (Kaufmann, 2009). In the lungs, increased alveolar macrophages, with pigment, occurred in both sexes exposed to 0.285, 1.41 or 5.60 mg/m3, with an exposure-related increase in severity. This finding was predominantly characterised by increased numbers of alveolar macrophages with a diffuse distribution. Black pigment was present within the cytoplasm of the macrophages. This reflected increased clearance of the test item. In addition, minimal bronchioloalveolar inflammation occurred in both sexes exposed to 5.60 mg/m3, which indicated a low level of damage at the bronchioloalveolar junction. These findings correlated with increased group mean absolute and adjusted lung and bronchi weight and generally correlated with abnormal (grey) coloration of the lung and bronchi in both sexes exposed to 5.60 mg/m3. Increased cellularity of the bronchial associated lymphoid tissue was also seen in some males and one female exposed to 5.60 mg/m3, and this was considered secondary to the inflammatory changes seen in bronchioloalveolar areas. In terms of adversity, the presence of diffusely distributed increased alveolar macrophages at a moderate severity and inflammatory changes were considered adverse in animals exposed to 5.60 mg/m3. There were no histopathological findings at 0.285 or 1.41 mg/m3 that were considered adverse. No treatment-related changes were observed at histological examination of the reproductive organs.

Under the experimental conditions, the inhalation administration of Graphistrength C100 at achieved exposure levels of 0.285, 1.41 or 5.60 mg/m3 was well tolerated. There was no evidence of any effects on mating performance or fertility of the adult animals or on the survival or development of the F1 offspring and Graphistrength C100 showed no evidence of being an endocrine disruptor. The no-observed-adverse-effect concentration (NOAEC) for systemic and reproductive/developmental toxicity was considered to be 5.60 mg/m3.

Microscopically in the lungs increased alveolar macrophages, with pigment, were seen in both sexes at all exposure levels, with an exposure-related increase in severity. At 5.60 mg/m3 this was accompanied by bronchioloalveolar inflammation and therefore the lung changes at 5.60 mg/m3 were considered as a normal physiological response to the overload of the lung with insoluble particles*. The no-observed-adverse-effect concentration (NOAEC) for local effects was considered to be 1.41 mg/m3.

 

*Kaufmann et al. (2009). Larynx Squamous Metaplasia: A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment. Experimental Toxicology Pathology, 61: 591-603.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Juillet 2017 to december 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: RccHan™ WIST
Details on species / strain selection:
The RccHanTM;WIST was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males 13 to 14 weeks old.
Females 14 to 15 weeks old
- Weight at study initiation:
Males 306 to 342 g.
Females 198 to 216 g.
- Fasting period before study: no
- Housing:
Pre-pairing up to five animals of one sex
Pairing one male and one female
Males after mating up to five animals
Gestation one female
Lactation one female + litter
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet
- Water (e.g. ad libitum): Potable water from the public supply
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: May to September 2017
Route of administration:
inhalation: dust
Type of inhalation exposure (if applicable):
nose only
Mass median aerodynamic diameter (MMAD):
> 2.4 - < 3 µm
Geometric standard deviation (GSD):
2.38
Remarks on MMAD:
it was not possible to assess particle size by impaction for Group 2.
The mass MMAD and Mean number MMAD (via WPS) was similar for Groups 2, 3 and 4 during each week of exposure and were in the range of 0.195 to 0.545 µm for mean Mass and 0.0231 to 0.0829 µm for mean number MMAD.
Vehicle:
clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow past snout only chamber. Aluminum alloy construction comprising a base unit, two animal exposure sections each with 16 exposure ports, a top section and a pre chamber.
- Method of holding animals in test chamber: Polycarbonate snout-only restraint tube. Animals fur was washed after removal from restraint tube.
- System of generating particulates/aerosols: Solid Aerosol Generator. The SAG technique for the dispersion of powders comprises of two steps, the continuous supply of test item to the disperser and the dispersal of the test item as an aerosol. The method for metering powder
to the disperser was to use a moving toothed belt. A regulated flow of compressed air conducted the aerosol produced to the in-line jet mill.
The jet mill utilizes streams of compressed air to causes high velocity particle-particle collisions. The collisions cause particles to lose mass. The jet mill was attached to a stainless steel elutriator.
The aerosol was conducted through plastic tubing from the elutriator to waste, an air operated vacuum pump (with an incorporated unobstructed venturi passage), incorporated into the plastic tubing, was used to supply the Group 4 chamber. An air operated vacuum pump, attached to the Group 4 extraction tubing conducted the diluted aerosol to the Group 3 chamber; similarly an air operated pump was attached to the Group 3 extraction tubing which conducted the diluted aerosol to the Group 2 chamber.
- Temperature in air chamber: 19.7-23.8°C
- Air flow rate:
. Generation Airflow
o Generator flow: 20 L/minute
o Jet mill flow: 20 + 20 + 40 L/minute
o Elutriator flow: 30 - 35 L/minute
o From in-house compressed air system – breathing quality
o Extract flow: 56 L/minute
o Drawn by in-house vacuum system and filtered locally
. System Airflows
o Venturi device flow: 15 L/minute (Group 2)
(Air operated vacuum pump) 10 – 14 L/minute (Group 3)
7 L/minute (Group 4)
o Diluent flow: 33 - 36 L/minute (Group 2)
6 - 12 L/minute (Group 3)
41 - 45 L/minute (Group 4)
o From in-house compressed air system – breathing quality
o Extraction flow: 55 - 56 L/minute (Group 2)
55 - 60 L/minute (Group 3)
70 L/minute (Group 4)
- Method of particle size determination:
Determined by cascade impaction
* samples collected as follows:
. Impactor type: Marple 290 Series (298 configuration)
. Collection media: Stainless steel substrates and Glass fiber final stage filter (Stainless steel substrates lightly lubricated with vacuum grease to aid sample collection)
. Sample flow: 3.0 L/minute
. Sample volume: Measured by wet-type gas meter
. Sample frequency: Minimum of 1 sample/week (Groups 3 and 4)
. Sample location: Animal exposure port
. Sample analysis: Gravimetric MMAD and GSD derived
No impaction particle size distribution samples were obtained from Group 2 due to the low target aerosol concentration and the high sample volume required to achieve a sample that could be weighed.

Particle Size Distribution (WPS)
* Samples collected as follows:
. Sample frequency: 1 sample/week/test group
. Sample location: Animal exposure port
* Recipe parameters:
. WPS model: 1000XP
. Instrument mode: WPS
. DMA channels: 96
. LPS: 24
. Time/DMA channel: 2 seconds
. Sample time: 192 seconds
. Wait time: 15 seconds
. Total sample time: 3 minutes 27 seconds
. Lower particle size: 10.0 nanometers
. Upper particle size: 10000 nanometers
- Treatment of exhaust air: Drawn by in-house vacuum system and filtered locally

TEST ATMOSPHERE
- Brief description of analytical method used:
Aerosol samples collected as follows:
. Filter type: Glass fiber filter, held in an open face filter holder
. Sample flow: 5.0 L/minute
. Sample volume: Measured by wet-type gas meter
. Sample frequency: 1 sample from Group 2/day (sample ran for 340 minutes, started at approximately 10 minutes into exposure)
Minimum of 3 samples from Group 3 and Group 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
¿ Sample location: Animal exposure port
¿ Sample analysis: Gravimetric
- Samples taken from breathing zone: yes
Details on mating procedure:
Pairing commenced: After two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: for a minimum of 4 weeks
Females: 2 weeks pre-pairing up to Day 19 post-coitum (pc). Untreated from Day 20 pc to Day 4 of lactation. Treatment reinstated Day 5 of lactation until Day 12.
Frequency of treatment:
6 hours, 7 days a week
Dose / conc.:
0.25 other: mg/m3 air (target conc.)
Remarks:
0.285 mg/m3 (analytical)
Dose / conc.:
1.25 other: mg/m3 air (target conc.)
Remarks:
1.41 mg/m3 (analytical)
Dose / conc.:
5 other: mg/m3 air (target conc.)
Remarks:
5.60 mg/m3 air (analytical)
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The exposure levels were selected on results of a 13 week inhalation study in rats (Harlan Study Number 48447) in which male and female RccHan™: WIST(SPF) rats were exposed to 0, 0.05, 0.25 and 5.0 mg/m3 of a respirable aerosol of Graphistrength™ C100 6 hours per day, 5 days per weeks. The effects were limited to a pulmonary inflammation at 5.0 mg/m3 characterized by changes in BALF parameters, alveolar granulocyte infiltration, bronchiolar cell hypertrophy/hyperplasia and interstitial inflammation. The NOAEC was 0.25 mg/m3.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Visually inspected at least twice daily for evidence of reaction to treatment or ill-health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week for all animals (females only to mating).
F0 females: Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation.

BODY WEIGHT: Yes
F0 males: Week -1 (or once during acclimatisation). Day that treatment commences. Each week. Before necropsy.
F0 females: Week -1 (or once during acclimatisation). Day that treatment commences and weekly until pairing. Days 0, 3, 7, 10, 14, 17 and 20 after mating and Days 1, 4 and 13 of lactation. Before necropsy.

FOOD CONSUMPTION: Yes
F0 males and females: Weekly before pairing (from first day of treatment until pairing).
F0 females: Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3, 4-6 and 7-12 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
- Thyroid hormone analysis
Thyroxine (T4) at termination on F0 males
Oestrous cyclicity (parental animals):
Dry smears Using cotton swabs on the following occasions:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to the study.
For 15 days before pairing.
For 4 days before scheduled termination (Days 10 to 13 of lactation).

Wet smears Using pipette lavage during the following phases:
After pairing until mating.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain on days 1, 4, 7 and 13 of age, physical or behavioural abnormalities, anogenital distance (AGD) on day 4 of age, nipple count (male pups) on day 12 of age, thyroxine (T4) on Day 13 of age. .
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals during Week 5 of respective treatment.
- Maternal animals:
F0 Females failing to mate on day 25 after last day of pairing.
F0 Females failing to produce viable litter on day 25 after mating.
F0 Females whose litters die before Day 13 on or after day last offspring dies.
Females killed at termination on day 13 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of[external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.

Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths All F0 animals from all groups. All specified in Table 1
Scheduled kill All F0 animals in Groups 1 and 4 All specified in Table 1
All F0 animals of Groups 2 and 3 Lung and abnormalities
All F0 males of Groups 2 and 3 Tracheobronchial lymph node
All F0 females of Groups 2 and 3 Larynx

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.

GROSS NECROPSY
- These animals were subjected to postmortem examinations as follows:
Examined externally, if found to be normal offspring discarded without further examination. Any externally abnormal offspring will also be examined internally. Attention given to external reproductive genitals. Abnormal tissues retained.
F1 offspring on Day 13: Thyroid gland retained from one male and one female in each litter. If insufficient numbers of Day 13 offspring were available for hormone sampling and thyroid retention/preservation, priority was given to hormone sampling

HISTOPATHOLOGY / ORGAN WEIGTHS: No
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including pre-coital interval and mating performance the similarity of the data was such that analyses were not considered to be necessary:
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during gestation and lactation
Estrous cycles
Pre-coital interval
Mating performance and fertility
Gestation length
Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index)
Ano-genital distance, adjusted for pup body weight
Organ weights, both absolute and adjusted for terminal body weight
Hormone analysis

The following comparisons were performed:
Group 1 vs 2, 3 and 4
Reproductive indices:
Percentage mating (%) = Number of animals mating x 100 /Animals paired
Conception rate (%) = Number of animals achieving pregnancy x 100/Animals mated
Fertility index (%) = Number of animals achieving pregnancy x 100/ Animals pairing
Gestation index (%) = Number of live litters born x 100/ Number pregnant
Offspring viability indices:
Post-implantation survival index (%) = Total number of offspring born x 100/ Total number of uterine implantation sites
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering x 100/ Total number of offspring born
Viability index (%) = Number of live offspring on Day 4 after littering x 100/ Number live offspring on Day 1
Lactation index (%) = Number of live offspring on Day 13 after littering x 100/ Number live offspring on Day 4
Percentage males = Number of males in litter x 100 / Total number of offspring in litter
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related clinical observations.
Black staining (head) was observed as a sign associated with dosing for all test groups, the incidence and persistence of this observation was related to exposure concentration. Wet fur was also observed, on occasion, post exposure. These signs were considered to be associated with the route and duration of administration and the physical properties of the test item (a black powder). Black staining was also noted during the detailed weekly physical examinations on the head and forelimbs.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No body weight gain was observed for females exposed to 5.60 mg/m3 compared with control over the first 2 weeks of exposures.
During the gestation phase body weight gains for treated females were similar to control, during the lactation phase lower body weight gains were observed for all treated female groups, as low as 0.66X control; however this finding lacked exposure relationship and is thus considered to be unrelated to the test item.
There were no effects on body weight gain for males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was slightly low for females exposed to 1.41 or 5.60 mg/m3 over the first 2 weeks of exposures compared with control (approx. 0.93X control). Food consumption during gestation was similar to control for all treated female groups but was slightly lower than control for females exposed to 1.41 or 5.60 mg/m3 during the last week of lactation (0.90X control).
There were no effects on food consumption for males.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals Killed After 4 Weeks of Treatment on Day 13 of Lactation
- Treatment Related Findings
Changes related to treatment with Graphistrength© C100 were seen in the larynx of females only, tracheobronchial lymph nodes of males only and lungs of both sexes.

Larynx
Minimal squamous metaplasia of the respiratory epithelium occurred in females exposed to 1.41 or 5.60 mg/m3, with an exposure-related increase in incidence.

Summary of treatment related findings in the larynx

Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Exposure Level (mg/m3) 0 0.285 1.41 5.60 0 0.285 1.41 5.60
Squamous Metaplasia, Respiratory Epithelium
Minimal 0 0 0 0 0 0 0 9
Total 0 0 0 0 0 0 3 9
Number of tissues examined 10 0 0 10 9 10 10 10

Lung
Increased alveolar macrophages, with pigment, and bronchioloalveolar inflammation occurred in all males and females exposed to 5.60 mg/m3. Increased alveolar macrophages, with pigment, was also seen in most males and one female exposed to 1.41 mg/m3. In addition, an increased incidence of increased cellularity of the bronchus-associated lymphoid tissue (BALT) was recorded in some males and one female exposed to 5.60 mg/m3, when compared to controls.

Summary of treatment related findings in the lung

Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Exposure Level (mg/ m3) 0 0.285 1.41 5.60 0 0.285 1.41 5.60
Increased Alveolar Macrophages, Pigment, Diffuse
Minimal 0 10 0 1 0 10 1 0
Slight 0 0 10 6 0 0 9 1
Moderate 0 0 0 3 0 0 0 9
Total 0 10 10 10 0 10 10 10
Inflammation, Bronchioloalveolar
Minimal 0 0 0 10 0 0 0 10
Total 0 0 0 10 0 0 0 10
Increased Cellularity, BALT
Minimal 1 0 0 6 0 0 0 1
Total 1 0 0 6 0 0 0 1
Number of tissues examined 10 10 10 10 9 10 10 10

- Incidental Findings
In the testes, minimal to slight tubular degeneration of the seminiferous epithelium, with a focal or multifocal distribution, and minimal to moderate luminal cell debris in the epididymis, were recorded in most control and treated males. In the testes, tubular degeneration was characterised by increased degeneration of round and elongated spermatids, with the formation of eosinophilic bodies in the tubular lumen. This finding showed no exposure level-relationship and no particular stage of the spermatogenic cycle was affected. In nose-only inhalation studies, degenerative testicular changes are sometimes seen at a higher frequency than other studies. This is likely to be due to stress during the restraint used during inhalation procedure, due to either heat or immobilization (Mowat et al., 2014). Therefore, these findings were considered to be procedural
All other findings were considered to be incidental and not related to treatment.

Description (incidence and severity):
Thyroid hormone sampling
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.
Mean T4 concentration for Day 13 female offspring from the groups exposed to 1.41 or 5.60 mg/m3 were slightly higher than the concentration from control offspring (up to 1.28X), however there was a large degree of variation in the individual values and all Day 13 of age male and female offspring had concentrations that were in the region of the endogenous levels observed in the control matrix used to prepare the QC samples, therefore this difference was considered to represent normal biological variation.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females assigned to this study had regular estrous cycles prior to the start of treatment. There were no treatment related findings on estrous cycles in the first 2 weeks of treatment prior to pairing. One female exposed to 5.60 mg/m3 (No. 131) was acyclic from the start of exposures, however this female returned to estrous and mated within 4 days of pairing.
(Examination also performed during the 90-day inhalation toxicity study (Broich, 2016): no effect)
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
Examinations performed during the 90-day inhalation toxicity study (Broich, 2016): no effect
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment related effects on mating performance or fertility.There were no test item related effects on mating performance or fertility.
The pre-coital interval was 1-4 days for all pairings, indicating mating occurred at the first estrous opportunity.
All pairings showed evidence of mating, and conception occurred in all but 1 control female.
The gestation length for all females was between 22 and 23 days. The gestation index was 100% in all groups.
Prior to termination all females smeared were in diestrus.
Dose descriptor:
NOAEC
Remarks:
Systemic toxicity
Effect level:
>= 5.6 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEC
Remarks:
Local effect
Effect level:
1.41 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
5.6 other: mg/m3 air (analytical)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weight change for male offspring from the 5.60 mg/m3 group was slightly higher than the control group (1.1X) but in the absence of similar effects in females and the small magnitude of the change, this was considered unrelated to the test item.
There were no effects on body weight for offspring from the 0.285 or 1.41 mg/m3 groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no effects on the number of implantations, litter size, sex ratio, survival indices or ano-genital distances.
Nipple counts on Day 13 were zero for all male offspring
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
5.6 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Inhalation administration of Graphistrength© C100 at achieved exposure levels of 0.285, 1.41 or 5.60 mg/m3 was well tolerated. There was no evidence of any effects on mating performance or fertility of the adult animals or on the survival or development of the F1 offspring and Graphistrength© C100 showed no evidence of being an endocrine disruptor. The no-observed-adverse-effect concentration (NOAEC) for systemic and reproductive/developmental toxicity was considered to be 5.60 mg/m3.
Microscopically in the lungs increased alveolar macrophages, with pigment, were seen in both sexes at all exposure levels, with an exposure-related increase in severity. At 5.60 mg/m3 this was accompanied by bronchioloalveolar inflammation and therefore the lung changes at 5.60 mg/m3 were considered adverse. The no-observed-adverse-effect concentration (NOAEC) for local toxicity was considered to be 1.41 mg/m3.
Executive summary:

In an OECD 421 study (Robinson, 2018), groups of 10 male and female Han Wistar rats were exposed nose-only by inhalation, 6 hours per day, 7 days per week, for 2 weeks pre-pairing up to Day 19 post-coitum (pc) (a minimum of 4 weeks for the males) and from Day 5 of lactation until Day 12 to a respirable aerosol of Graphistrength C100 at actual concentrations of 0, 0.285, 1.41 and 5.60 mg/m3 (MMAD 2.4-3.0 µm, GSD 2.38). The animals were checked twice daily for mortality, morbidity and clinical signs during the treatment period. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 3, 7, 10, 14 and 20 post-coitum (p.c.)and 1-3, 4-6 and 7-12 of lactation. Estrous cyclicity was checked for 15 days before pairing. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1, 4, 7 and 13 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 4 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p. Litter standardization was performed on Day 4 p.p.in order to reduce the litter size-induced variability in pup growth and development. Thyroid hormone (TSH, T3 and T4) plasma levels were determined on the day of sacrifice in parental males and in Day 13 p.p. pups. Final body weights and selected organs weights (lungs, ovaries and male reproductive organs) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on larynx, lungs, tracheobronchial lymph nodes in all groups and on nasal passages, ovaries (with oviducts), epididymides and/or testes, pituitary in the control and high-dose groups, and on all macroscopic lesions. A macroscopic post-mortem examination was performed on pups on Day 14 p.p., including those found dead before study termination.

Test item (black) staining on the head was recorded as a sign associated with dosing for all test groups, the incidence of this observation was related to exposure concentration. Wet fur seen on occasion is considered to be associated with the method of restraint. Test item (black) staining was also noted during the detailed weekly physical examinations on the head and forelimbs. No body weight gain was observed for females exposed to 5.60 mg/m3 compared with control over the first 2 weeks of exposures. During the gestation phase body weight gains for treated females were similar to control, during the lactation phase lower body weight gains were observed for all treated female groups, as low as 0.66X control; however this finding lacked exposure relationship and is thus considered to be unrelated to the test item. There were no effects on body weight gain for males. Food consumption was slightly low for females exposed to 1.41 or 5.60 mg/m3 over the first 2 weeks of exposures compared with control (approx. 0.9X control). Food consumption during gestation was similar to control for all treated female groups but was slightly lower than control for females exposed to 1.41 or 5.60 mg/m3 during lactation (0.9X control). There were no effects on food consumption for males. There were no test item related effects on mating performance, estrous cycles, pre-coital Interval, fertility and gestation length, and estrous stage at termination. All thyroid hormone sampling were completed, T4 results from Day 13 pups and adult males are pending. There were no effects on the number of implantations, litter size, sex ratio, survival indices or ano-genital distances. Group mean body weight change for male offspring from the 5.60 mg/m3 group was slightly higher than the control group (1.1X) but in the absence of similar effects in females and the small magnitude of the change, was considered unrelated to the test item. Nipple counts on Day 13 were zero for all male offspring. Group mean lung and bronchi weight (unadjusted and adjusted) were higher than control for both sexes exposed to 1.41 or 5.60 mg/m3 , up to 1.3X for adjusted weights, for males and females (exposure related). Abnormal color (grey) was seen in the lungs of all animals exposed to 5.60 mg/m3 and in the majority of animals exposed to 1.41 mg/m3, in both sexes. Abnormal color (grey) was seen in the tracheobronchial lymph nodes of all females exposed to 5.60 mg/m3 and the majority of females exposed to 1.41 mg/m3. Dark lymph nodes were noted in some males exposed to 1.41 or 5.60 mg/m3. Enlargement was seen in the majority of males exposed to 5.60 mg/m3 and several males exposed to 1.41 mg/m3. Changes related to inhalation administration of the test item were seen in the larynx of females only and lungs of both sexes. In the larynx, minimal squamous metaplasia occurred in treated females, with an exposure- related increase in incidence. This finding was characterised by a focal loss of cilia and focal flattening of the surface epithelial cells, primarily at the basis of the epiglottis. This change is indicative of low grade irritation of the epithelium of the ventral larynx and is considered non-adverse. The rat larynx is highly sensitive to irritant materials due to species-specific anatomical, airflow and histological features, and as such squamous metaplasia in this region is generally considered irrelevant to man (Kaufmann, 2009). In the lungs, increased alveolar macrophages, with pigment, occurred in both sexes exposed to 0.285, 1.41 or 5.60 mg/m3, with an exposure-related increase in severity. This finding was predominantly characterised by increased numbers of alveolar macrophages with a diffuse distribution. Black pigment was present within the cytoplasm of the macrophages. This reflected increased clearance of the test item. In addition, minimal bronchioloalveolar inflammation occurred in both sexes exposed to 5.60 mg/m3, which indicated a low level of damage at the bronchioloalveolar junction. These findings correlated with increased group mean absolute and adjusted lung and bronchi weight and generally correlated with abnormal (grey) coloration of the lung and bronchi in both sexes exposed to 5.60 mg/m3. Increased cellularity of the bronchial associated lymphoid tissue was also seen in some males and one female exposed to 5.60 mg/m3, and this was considered secondary to the inflammatory changes seen in bronchioloalveolar areas. In terms of adversity, the presence of diffusely distributed increased alveolar macrophages at a moderate severity and inflammatory changes were considered adverse in animals exposed to 5.60 mg/m3. There were no histopathological findings at 0.285 or 1.41 mg/m3 that were considered adverse. No treatment-related changes were observed at histological examination of the reproductive organs.

Under the experimental conditions, the inhalation administration of Graphistrength C100 at achieved exposure levels of 0.285, 1.41 or 5.60 mg/m3was well tolerated. There was no evidence of any effects on mating performance or fertility of the adult animals or on the survival or development of the F1 offspring and Graphistrength C100 showed no evidence of being an endocrine disruptor. The no-observed-adverse-effect concentration (NOAEC) for systemic and reproductive/developmental toxicity was considered to be 5.60 mg/m3.

Microscopically in the lungs increased alveolar macrophages, with pigment, were seen in both sexes at all exposure levels, with an exposure-related increase in severity. At 5.60 mg/m3 this was accompanied by bronchioloalveolar inflammation and therefore the lung changes at 5.60 mg/m3 were considered adverse. The no-observed-adverse-effect concentration (NOAEC) for local toxicity was considered to be 1.41 mg/m3.

 

Kaufmann et al. (2009). Larynx Squamous Metaplasia: A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment. Experimental Toxicology Pathology, 61: 591-603.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
5.6 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Key study
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

In a 90-day inhalation toxicity study performed according to the OECD TG #413 with Graphistrength C100 (Broich, 2016), no histological alteration of the male and female reproductive organs, no change in the oestrous cycle and no modification of the sperm parameters has been observed up to the exposure concentration of 4.84 mg/m3.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day)
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
See RSS in section 7.5.2.
Route of administration:
inhalation: dust
Type of inhalation exposure (if applicable):
nose only
Vehicle:
clean air
Details on exposure:
See RSS in section 7.5.2.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See RSS in section 7.5.2.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.05 other: mg/m³ air (target)
Remarks:
0.059 mg/m³ air (analytical)
Dose / conc.:
0.25 other: mg/m³ air (target)
Remarks:
0.279 mg/m³ air (analytical)
Dose / conc.:
5 other: mg/m³ air (target)
Remarks:
4.84 mg/m³ air (analytical)
No. of animals per sex per dose:
35
Control animals:
yes, sham-exposed
Details on study design:
See RSS in section 7.5.2.
Statistics:
See RSS in section 7.5.2.
Dose descriptor:
NOAEC
Effect level:
> 4.84 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: No treatment related effects on oestrous cycle, sperm parameters, and male and female reproductive organs
See RSS in section 7.5.2.
Executive summary:

See RSS in section 7.5.2.

Justification for classification or non-classification

According to the available data and Regulation (EC) No 1272/2008, no classification is required for reproductive and developmental toxicity.

Additional information